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Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

Li S, Payne S, Wang F, Claus P, Su Z, Groth J, Geradts J, de Ridder G, Alvarez R, Marcom PK, Pizzo SV, Bachelder RE - Breast Cancer Res. (2015)

Bottom Line: The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies.Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance.Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, N.C., 27710, USA. shenduo.li@duke.edu.

ABSTRACT

Introduction: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].

Methods: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.

Results: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.

Conclusion: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

No MeSH data available.


Related in: MedlinePlus

Chemo-residual triple-negative (TN) breast tumor cells emanating from a short-term chemotherapy treatment model do not exhibit cancer stem-like cell behavior. a In vitro model of TN breast cancer chemo-resistance. SUM159 and BT549 tumor cells were treated with doxorubicin (Dox) (1 μg/ml, 0.5 μg/ml, respectively) for 2 days, after which chemotherapy was removed and fresh medium was added. Between 7 and 10 days, a small number of chemo-residual cells (0.1 % of the original population) remained, and exhibited reduced proliferation compared to parental (untreated) cells. Approximately 2 weeks after chemotherapy withdrawal, chemo-residual cells resumed proliferation and established colonies. Pictures of parental (untreated) cells, chemo-residual cells, and colonies emanating from chemo-residual cells were taken on days 0, 7, 14 (SUM159 cells) or days 0, 10, 18 (BT549 cells), respectively. Magnification ×200. Similar data were obtained by treating SUM159 cells with docetaxel (100 nM) for 2 days [8]. b SUM159 cells were treated with Dox as described in Fig. 1a. Parental and chemo-residual cells were seeded at equal density in a 96-well plate. Left panel: proliferation was determined by 3H-thymidine incorporation. Right panel: cell viability was assessed by alamar blue. Error bars represent SD, n = 6, ***p <0.001, two-tailed Student’s t test. c SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were seeded at the indicated numbers into a mammosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. d Parental and chemo-residual cells harvested on day 8 after docetaxel treatment were seeded into a mammmosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. e RNA was extracted from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment. Equivalent amounts were subjected to hypoxia inducible factor-1α (HIF-1α) real-time PCR. Results are expressed as the mean HIF-1α/β- actin ratio from triplicate wells (+/- SD). **p <0.01, two-tailed Student’s t test. f Total cellular extracts obtained from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were subjected to SDS-PAGE and immunoblotted with a HIF-1α or β-actin antibody, followed by secondary antibody
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Fig1: Chemo-residual triple-negative (TN) breast tumor cells emanating from a short-term chemotherapy treatment model do not exhibit cancer stem-like cell behavior. a In vitro model of TN breast cancer chemo-resistance. SUM159 and BT549 tumor cells were treated with doxorubicin (Dox) (1 μg/ml, 0.5 μg/ml, respectively) for 2 days, after which chemotherapy was removed and fresh medium was added. Between 7 and 10 days, a small number of chemo-residual cells (0.1 % of the original population) remained, and exhibited reduced proliferation compared to parental (untreated) cells. Approximately 2 weeks after chemotherapy withdrawal, chemo-residual cells resumed proliferation and established colonies. Pictures of parental (untreated) cells, chemo-residual cells, and colonies emanating from chemo-residual cells were taken on days 0, 7, 14 (SUM159 cells) or days 0, 10, 18 (BT549 cells), respectively. Magnification ×200. Similar data were obtained by treating SUM159 cells with docetaxel (100 nM) for 2 days [8]. b SUM159 cells were treated with Dox as described in Fig. 1a. Parental and chemo-residual cells were seeded at equal density in a 96-well plate. Left panel: proliferation was determined by 3H-thymidine incorporation. Right panel: cell viability was assessed by alamar blue. Error bars represent SD, n = 6, ***p <0.001, two-tailed Student’s t test. c SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were seeded at the indicated numbers into a mammosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. d Parental and chemo-residual cells harvested on day 8 after docetaxel treatment were seeded into a mammmosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. e RNA was extracted from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment. Equivalent amounts were subjected to hypoxia inducible factor-1α (HIF-1α) real-time PCR. Results are expressed as the mean HIF-1α/β- actin ratio from triplicate wells (+/- SD). **p <0.01, two-tailed Student’s t test. f Total cellular extracts obtained from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were subjected to SDS-PAGE and immunoblotted with a HIF-1α or β-actin antibody, followed by secondary antibody

Mentions: To enrich for chemo-resistant tumor cells, we treated SUM159 and BT549 TN breast tumor cells with doxorubicin at a clinically relevant concentration [26]. Doxorubicin was removed after 2 days, and fresh medium was added. Although the vast majority of cells were eliminated by day 6, we observed a small number of residual, viable tumor cells, representing 0.1 % of the original population, on day 7 post treatment (Fig. 1a). These chemo-residual cells were metabolically active, but exhibited significantly reduced proliferation (Fig. 1b). Chemo-residual tumor cells resumed proliferation approximately 2 weeks post chemotherapy treatment. Our previous studies show that colonies from this model continue to expand after 18 days, and exhibit resistance to multiple classes of chemotherapy for as long as 50 days [8].Fig. 1


Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

Li S, Payne S, Wang F, Claus P, Su Z, Groth J, Geradts J, de Ridder G, Alvarez R, Marcom PK, Pizzo SV, Bachelder RE - Breast Cancer Res. (2015)

Chemo-residual triple-negative (TN) breast tumor cells emanating from a short-term chemotherapy treatment model do not exhibit cancer stem-like cell behavior. a In vitro model of TN breast cancer chemo-resistance. SUM159 and BT549 tumor cells were treated with doxorubicin (Dox) (1 μg/ml, 0.5 μg/ml, respectively) for 2 days, after which chemotherapy was removed and fresh medium was added. Between 7 and 10 days, a small number of chemo-residual cells (0.1 % of the original population) remained, and exhibited reduced proliferation compared to parental (untreated) cells. Approximately 2 weeks after chemotherapy withdrawal, chemo-residual cells resumed proliferation and established colonies. Pictures of parental (untreated) cells, chemo-residual cells, and colonies emanating from chemo-residual cells were taken on days 0, 7, 14 (SUM159 cells) or days 0, 10, 18 (BT549 cells), respectively. Magnification ×200. Similar data were obtained by treating SUM159 cells with docetaxel (100 nM) for 2 days [8]. b SUM159 cells were treated with Dox as described in Fig. 1a. Parental and chemo-residual cells were seeded at equal density in a 96-well plate. Left panel: proliferation was determined by 3H-thymidine incorporation. Right panel: cell viability was assessed by alamar blue. Error bars represent SD, n = 6, ***p <0.001, two-tailed Student’s t test. c SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were seeded at the indicated numbers into a mammosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. d Parental and chemo-residual cells harvested on day 8 after docetaxel treatment were seeded into a mammmosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. e RNA was extracted from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment. Equivalent amounts were subjected to hypoxia inducible factor-1α (HIF-1α) real-time PCR. Results are expressed as the mean HIF-1α/β- actin ratio from triplicate wells (+/- SD). **p <0.01, two-tailed Student’s t test. f Total cellular extracts obtained from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were subjected to SDS-PAGE and immunoblotted with a HIF-1α or β-actin antibody, followed by secondary antibody
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4491247&req=5

Fig1: Chemo-residual triple-negative (TN) breast tumor cells emanating from a short-term chemotherapy treatment model do not exhibit cancer stem-like cell behavior. a In vitro model of TN breast cancer chemo-resistance. SUM159 and BT549 tumor cells were treated with doxorubicin (Dox) (1 μg/ml, 0.5 μg/ml, respectively) for 2 days, after which chemotherapy was removed and fresh medium was added. Between 7 and 10 days, a small number of chemo-residual cells (0.1 % of the original population) remained, and exhibited reduced proliferation compared to parental (untreated) cells. Approximately 2 weeks after chemotherapy withdrawal, chemo-residual cells resumed proliferation and established colonies. Pictures of parental (untreated) cells, chemo-residual cells, and colonies emanating from chemo-residual cells were taken on days 0, 7, 14 (SUM159 cells) or days 0, 10, 18 (BT549 cells), respectively. Magnification ×200. Similar data were obtained by treating SUM159 cells with docetaxel (100 nM) for 2 days [8]. b SUM159 cells were treated with Dox as described in Fig. 1a. Parental and chemo-residual cells were seeded at equal density in a 96-well plate. Left panel: proliferation was determined by 3H-thymidine incorporation. Right panel: cell viability was assessed by alamar blue. Error bars represent SD, n = 6, ***p <0.001, two-tailed Student’s t test. c SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were seeded at the indicated numbers into a mammosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. d Parental and chemo-residual cells harvested on day 8 after docetaxel treatment were seeded into a mammmosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. e RNA was extracted from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment. Equivalent amounts were subjected to hypoxia inducible factor-1α (HIF-1α) real-time PCR. Results are expressed as the mean HIF-1α/β- actin ratio from triplicate wells (+/- SD). **p <0.01, two-tailed Student’s t test. f Total cellular extracts obtained from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were subjected to SDS-PAGE and immunoblotted with a HIF-1α or β-actin antibody, followed by secondary antibody
Mentions: To enrich for chemo-resistant tumor cells, we treated SUM159 and BT549 TN breast tumor cells with doxorubicin at a clinically relevant concentration [26]. Doxorubicin was removed after 2 days, and fresh medium was added. Although the vast majority of cells were eliminated by day 6, we observed a small number of residual, viable tumor cells, representing 0.1 % of the original population, on day 7 post treatment (Fig. 1a). These chemo-residual cells were metabolically active, but exhibited significantly reduced proliferation (Fig. 1b). Chemo-residual tumor cells resumed proliferation approximately 2 weeks post chemotherapy treatment. Our previous studies show that colonies from this model continue to expand after 18 days, and exhibit resistance to multiple classes of chemotherapy for as long as 50 days [8].Fig. 1

Bottom Line: The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies.Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance.Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, N.C., 27710, USA. shenduo.li@duke.edu.

ABSTRACT

Introduction: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].

Methods: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.

Results: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.

Conclusion: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

No MeSH data available.


Related in: MedlinePlus